Alterations in well-regulated antioxidant enzyme reactions cause irreversible damage to cells and tissues. We recently reported that Cu,Zn-superoxide dismutase (Cu,ZnSOD) behaves as an enzyme that catalyzes the formation of free radicals using anionic scavengers and excess hydrogen peroxide as substrates. We report here the results of our studies using EPR spectroscopy and the spin trapping method with 5,5- dimethyl-1-pyrroline N-oxide (DMPO). We found that glutathione (GSH) and the thiol-specific antioxidant (TSA) enzyme are scavengers of oxygen and thiyl radicals, respectively. We also showed that alpha-ketoaldehyde, an oxidation product from amino acids, induces crosslinking of Cu,Zn-SOD. Thiyl radicals from dithiothreitol (DTT) and GSH were produced by horseradish peroxidase/hydrogen peroxide under aerobic and anaerobic conditions, and by the Fe(III)/oxygen system. The formation of DMPO- thiyl radical adducts was inhibited by TSA independently of the thiyl radical-generating conditions used. Site-directed mutagenesis of TSA revealed that cysteine 47 is required for thiyl radical removal. In addition, thiyl radicals react with oxygen to generate unidentified thiylperoxy species. Fe-EDTA reacts with this species to generate another reactive radical, which can abstract a hydrogen atom from ethanol. This reactive thiyl-oxygen radical is believed to be responsible for causing deleterious effects on biomolecules. Together, our data indicate that TSA protects biomolecules from oxidative damage by catalyzing the removal of thiyl radicals before they generate more reactive radicals. We have also found oxidative stress by hydrogen peroxide induces the formation of glutathionyl radicals inside NCB-20 cells in vivo without external addition of GSH. The signal intensity of glutathionyl radical was constant for one hour. This may indicate that TSA in the cell maintains the concentration of GSH by regenerating it from glutathionyl radicals.